Two isofunctional nitric oxide reductases in Alcaligenes eutrophus H16.

Two genes, norB and norZ, encoding two independent nitric oxide reductases have been identified in Alcaligenes eutrophus H16. norB and norZ predict polypeptides of 84.5 kDa with amino acid sequence identity of 90%. While norB resides on the megaplasmid pHG1, the norZ gene is located on a chromosomal DNA fragment. Amino acid sequence analysis suggests that norB and norZ encode integral membrane proteins composed of 14 membrane-spanning helices. The region encompassing helices 3 to 14 shows similarity to the NorB subunit of common bacterial nitric oxide reductases, including the positions of six strictly conserved histidine residues. Unlike the Nor enzymes characterized so far from denitrifying bacteria, NorB and NorZ of A. eutrophus contain an amino-terminal extension which may form two additional helices connected by a hydrophilic loop of 203 amino acids. The presence of a NorB/NorZ-like protein was predicted from the genome sequence of the cyanobacterium Synechocystis sp. strain PCC6803. While the common NorB of denitrifying bacteria is associated with a second cytochrome c subunit, encoded by the neighboring gene norC, the nor loci of A. eutrophus and Synechocystis lack adjacent norC homologs. The physiological roles of norB and norZ in A. eutrophus were investigated with mutants disrupted in the two genes. Mutants bearing single-site deletions in norB or norZ were affected neither in aerobic nor in anaerobic growth with nitrate or nitrite as the terminal electron acceptor. Inactivation of both norB and norZ was lethal to the cells under anaerobic growth conditions. Anaerobic growth was restored in the double mutant by introducing either norB or norZ on a broad-host-range plasmid. These results show that the norB and norZ gene products are isofunctional and instrumental in denitrification.     

Erwinia chrysanthemi tolC is involved in resistance to antimicrobial plant chemicals and is essential for phytopathogenesis

TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens. We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16. The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue. The E. chrysanthemi gene was able to functionally complement the E. coli tolC gene with respect to its role in MDR efflux pumps. A tolC mutant of E. chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals. This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised. The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical. These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E. chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue.     

Second-site suppressor mutations for the serine 202 to phenylalanine substitution within the interdomain loop of the tetracycline efflux protein Tet(C)

The serine 202 to phenylalanine substitution within the cytoplasmic interdomain loop of Tet(C) greatly reduces tetracycline resistance and efflux activity (Saraceni-Richards, C. A., and Levy, S. B. (2000) J. Biol. Chem. 275, 6101-6106). Second-site suppressor mutations were identified following hydroxylamine and nitrosoguanidine mutagenesis. Three mutations, L11F in transmembrane 1 (TM1), A213T in the central interdomain loop, and A270V in cytoplasmic loop 8-9, restored a wild type level of resistance and an active efflux activity in Escherichia coli cells bearing the mutant tet(C) gene. The Tet S202F protein with the additional A270V mutation was expressed in amounts comparable with the original mutant, whereas L11F and A213T Tet(C) protein mutants were overexpressed. Introduction of each single mutation into the wild type tet(C) gene by site-directed mutagenesis did not alter tetracycline resistance or efflux activity. These secondary mutations may restore resistance by promoting a conformational change in the protein to accommodate the S202F mutation. The data demonstrate an interaction of the interdomain loop with other distant regions of the protein and support a role of the interdomain loop in mediating tetracycline resistance.     

Bacterial multidrug efflux pumps: Mechanisms,physiology and pharmacological exploitations

Multidrug resistance (MDR) refers to the capability of bacterial pathogens to withstand lethal doses of structurally diverse drugs which are capable of eradicating non-resistant strains. MDR has been identified as a major threat to the public health of human being by the World Health Organization (WHO). Among the four general mechanisms that cause antibiotic resistance including target alteration, drug inactivation, decreased permeability and increased efflux, drug extrusion by the multidrug efflux pumps serves as an important mechanism of MDR. Efflux pumps not only can expel a broad range of antibiotics owing to their poly-substrate specificity, but also drive the acquisition of additional resistance mechanisms by lowering intracellular antibiotic concentration and promoting mutation accumulation. Over-expression of multidrug efflux pumps have been increasingly found to be associated with clinically relevant drug resistance. On the other hand, accumulating evidence has suggested that efflux pumps also have physiological functions in bacteria and their expression is subject tight regulation in response to various of environmental and physiological signals. A comprehensive understanding of the mechanisms of drug extrusion, and regulation and physiological functions of efflux pumps is essential for the development of anti-resistance interventions. In this review, we summarize the development of these research areas in the recent decades and present the pharmacological exploitation of efflux pump inhibitors as a promising anti-drug resistance intervention.     

Bacterial resistance modifying agents from Lycopus europaeus

As part of an ongoing project to identify plant natural products which modulate bacterial multidrug resistance (MDR), bioassay-guided isolation of an extract of Lycopus europaeus yielded two new isopimarane diterpenes, namely methyl-1alpha-acetoxy-7alpha 14alpha-dihydroxy-8,15-isopimaradien-18-oate (1) and methyl-1alpha,14alpha-diacetoxy-7alpha-hydroxy-8,15-isopimaradien-18-oate (2). The structures were established by spectroscopic methods. These compounds and several known diterpenes were tested for in vitro antibacterial and resistance modifying activity against strains of Staphylococcus aureus possessing the Tet(K), Msr(A), and Nor(A) multidrug resistance efflux mechanisms. At 512 microg/ml none of the compounds displayed any antibacterial activity but individually in combination with tetracycline and erythromycin, a two-fold potentiation of the activities of these antibiotics was observed against two strains of S. aureus that were highly resistant to these agents due to the presence of the multidrug efflux mechanisms Tet(K) (tetracycline resistance) and Msr(A) (macrolide resistance).     

Synthesis and evaluation of PSSRI-based inhibitors of Staphylococcus aureus multidrug efflux pumps

Phenylpiperidine selective serotonin reuptake inhibitors (PSSRIs) block the function of selected multidrug efflux pumps of Staphylococcus aureus. In this study PSSRI-based piperidine derivatives were prepared, evaluated for inhibition of two multidrug resistance (MDR)-conferring efflux pump systems, and tested for potentiation of antimicrobial activity of antibacterial efflux pump substrates. It is demonstrated that the 4-phenyl moiety of PSSRI-based efflux pump inhibitors (EPIs) is not an absolute structural requirement for inhibiting the NorA and MepA MDR efflux pumps. Potency of efflux inhibition is maintained or enhanced by replacing the aryloxymethyl substituent at position-3 of PSSRIs with arylalkene and arylthioether moieties. Novel 3-aryl piperidine EPIs that significantly increase substrate antibiotic activity against strains of S. aureus expressing NorA and MepA are described.     

Bacterial efflux systems and efflux pumps inhibitors

It is now well established that bacterial resistance to antibiotics has become a serious problem of public health that concerns almost all antibacterial agents and that manifests in all fields of their application. Among the three main mechanisms involved in bacterial resistance (target modification, antibiotic inactivation or default of its accumulation within the cell), efflux pumps, responsible for the extrusion of the antibiotic outside the cell, have recently received a particular attention. Actually, these systems, classified into five families, can confer resistance to a specific class of antibiotics or to a large number of drugs, thus conferring a multi-drug resistance (MDR) phenotype to bacteria. To face this issue, it is urgent to find new molecules active against resistant bacteria. Among the strategies employed, the search for inhibitors of resistance mechanisms seems to be attractive because such molecules could restore antibiotic activity. In the case of efflux systems, efflux pump inhibitors (EPIs) are expected to block the pumps and such EPIs, if active against MDR pumps, would be of great interest. This review will focus on the families of bacterial efflux systems conferring drug resistance, and on the EPIs that have been identified to restore antibiotic activity.     

Overproduction and purification of the Tn10-specified inner membrane tetracycline resistance protein Tet using fusions to β-galactosidase

Tetracycline resistance in the Enterobacteriaceae is mediated by a number of genetically related, usually plasmid-borne, determinants which specify an efflux system involving an inner membrane protein, Tet. Attempts to overproduce the Tn10 (Class B)-encoded Tet in Escherichia coli by cloning the structural gene tet downstream of the lambda PL promoter under regulation by temperature-sensitive lambda repressor cI857 were unsuccessful; induction at 42 degrees C resulted in filamentous, non-viable cells containing little detectable overproduction of the protein. However, cells containing tet fused to lacZ were resistant to tetracycline at 30 degrees C and synthesized modest amounts of a large fusion protein when induced at 42 degrees C. Fusion of the N-terminal half or the first 38 amino acids of tet to lacZ did lead to increased production of fusion proteins. Fusions could be purified by size or by LacZ immunoaffinity or substrate-affinity chromatography. In the latter method, selected detergents were required to counteract nonspecific binding of Tet to the adsorbant. Amino acid sequencing of the N-terminus of Tet-LacZ fusion proteins indicated that most molecules were blocked at this terminus. The sequence of an unblocked subpopulation was consistent with that expected from the nucleotide sequence. A collagen peptide linker, genetically placed between tet and lacZ, allowed recovery of purified Tet protein after collagenase treatment of the purified fusion protein.     

Stress impact of liposomes loaded with ciprofloxacin on the expression level of MepA and NorB efflux pumps of methicillin-resistant Staphylococcus aureus

International Microbiology - One mechanism of ciprofloxacin resistance is attributed to chromosomal DNA-encoded efflux pumps such as the MepA and NorB proteins. The goal of this research is to find...     

Development, validation, and application of PCR primers for detection of tetracycline efflux genes of gram-negative bacteria

Phylogenetic analysis of tetracycline resistance genes, which confer resistance due to the efflux of tetracycline from the cell catalyzed by drug:H(+) antiport and share a common structure with 12 transmembrane segments (12-TMS), suggested the monophyletic origin of these genes. With a high degree of confidence, this tet subcluster unifies 11 genes encoding tet efflux pumps and includes tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(J), tet(Y), tet(Z), and tet(30). Phylogeny-aided alignments were used to design a set of PCR primers for detection, retrieval, and sequence analysis of the corresponding gene fragments from a variety of bacterial and environmental sources. After rigorous validation with the characterized control tet templates, this primer set was used to determine the genotype of the corresponding tetracycline resistance genes in total DNA of swine feed and feces and in the lagoons and groundwater underlying two large swine production facilities known to be impacted by waste seepage. The compounded tet fingerprint of animal feed was found to be tetCDEHZ, while the corresponding fingerprint of total intestinal microbiota was tetBCGHYZ. Interestingly, the tet fingerprints in geographically distant waste lagoons were identical (tetBCEHYZ) and were similar to the fecal fingerprint at the third location mentioned above. Despite the sporadic detection of chlortetracycline in waste lagoons, no auxiliary diversity of tet genes in comparison with the fecal diversity could be detected, suggesting that the tet pool is generated mainly in the gut of tetracycline-fed animals, with a negligible contribution from selection imposed by tetracycline that is released into the environment. The tet efflux genes were found to be percolating into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. With yet another family of tet genes, this study confirmed our earlier findings that the antibiotic resistance gene pool generated in animal production systems may be mobile and persistent in the environment with the potential to enter the food chain.     

Gene duplication in the evolution of the two complementing domains of gram-negative bacterial tetracycline efflux proteins

The resistance of Gram- bacteria to the broad-spectrum antibiotic tetracycline (Tc) results from energy-dependent drug efflux mediated by the tet gene product, the cytoplasmic membrane Tet protein. Amino acid (aa) sequences deduced from total tet nucleotide sequences of three different resistance determinants (classes A, B and C) indicate that the protein products [Tet(A), Tet(B), and Tet(C)] share a common ancestor. Hydropathic analysis of Tet sequences predicts twelve transmembrane segments in each protein, with six occurring in each half of the molecule. More importantly, the linear distributions of these segments in the N- and C-terminal halves are nearly identical, suggesting that the two halves of each Tet protein are related by a process of tandem gene duplication and divergence. Indeed, a variable but significant conservation of sequence was detected among the N- and C-terminal halves for all possible comparisons of the three proteins. Such conservation was not observed within other prokaryotic integral membrane proteins or when other prokaryotic proteins were compared to Tet halves. Similarity, both in sequence and in predicted transmembrane structural organization, strongly suggests that a common ancestor of Tet(A), Tet(B), and Tet(C) arose by duplication of a gene reading frame specifying a transmembrane protein of approximately 200 aa residues. The two halves of Tet proteins correspond to the two domains, alpha and beta, which have distinct, complementary roles in Tc efflux. Nevertheless, selective constraints to function in the cytoplasmic membrane have apparently led to maintenance of similar patterns of secondary structural organization in these complementary domains.     

Coordinate overexpression of two RND efflux systems,ParXY and TtgABC,is responsible for multidrug resistance in Pseudomonas putida

Resistance Nodulation cell Division (RND) efflux pumps are known to contribute to the tolerance of Pseudomonas putida to aromatic hydrocarbons, but their role in antibiotic resistance has not been fully elucidated. In this study, two types of single-step multidrug-resistant (MDR) mutants were selected in vitro from reference strain KT2440. Mutants of the first type were more resistant to fluoroquinolones and β-lactams except imipenem, and overproduced the efflux system TtgABC as a result of mutations occurring in regulator TtgR. In addition to TtgABC, mutants of the second type such as HPG-5 were found to upregulate a novel RND pump, dubbed ParXY/TtgC, which accommodates cefepim, fluoroquinolones and aminoglycosides. As demonstrated by gene deletion experiments, TtgABC and ParXY/TtgC are both under the positive control of a two-component system, PpeRS. Whole-genome sequence analyses revealed that mutant HPG-5 harbours a mutation inactivating the gene (sucD) of succinyl-CoA synthetase, an enzyme of the tricarboxylic cycle. Disruption of sucD in strain KT2440 reproduced the resistance phenotype of HPG-5, and activated the glyoxylate shunt. Finally, identification of two MDR clinical strains of P. putida that jointly overexpress TtgABC and ParXY/TtgC, of which one is a sucD mutant, highlights the role of these efflux systems as determinants of antibiotic resistance.